Transcriptional programming of social hierarchy.

In this issue of Neuron, Choi and colleagues1 uncover the direct role of the transcription factor Pou3f1 in regulating dominance hierarchy in mice. Pou3f1 accomplishes this role via its action in specific prefrontal projection neurons that regulate behaviors associated with low social status.

A transcriptomic axis predicts state modulation of cortical interneurons.

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes1-6, but it is not known whether these subtypes have correspondingly diverse patterns of activity in the living brain. Here we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, which are organized by a single factor: position along the main axis of transcriptomic variation. We combined in vivo two-photon calcium imaging of mouse V1 with a transcriptomic method to identify mRNA for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 subclasses, 11 types and 35 subtypes using previously defined transcriptomic clusters3. Responses to visual stimuli differed significantly only between subclasses, with cells in the Sncg subclass uniformly suppressed, and cells in the other subclasses predominantly excited. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory subtypes that fired more in resting, oscillatory brain states had a smaller fraction of their axonal projections in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro7, and expressed more inhibitory cholinergic receptors. Subtypes that fired more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 subtypes shape state-dependent cortical processing.

Parenting as a model for behavioural switches.

Adaptability to ethologically relevant cues is fundamental for social interactions. As such, reproductive success relies on the ability of an animal to transition between parental and nonparental states. Though driven by genetically pre-programmed circuits, these instinctive repertoires are reshaped by internal state and experience, making parenting a robust model for the study of behavioural flexibility. As a functional wiring diagram for parenting emerges in mice, we are well placed to identify neural substrates and posit associated mechanisms underlying caregiving transitions. In this review, we discuss the importance of comprehensively characterising behaviour, highlight the role of shared circuit elements for behavioural malleability and explore plastic mechanisms that might guide switches between parental and nonparental repertoires.

Single-cell molecular and developmental perspectives of sexually dimorphic circuits underlying innate social behaviors.

While single-cell transcriptomics in the brain has uncovered a vast diversity of neural cell types in unprecedented detail, it is becoming increasingly urgent to address what exactly their functional roles are in the context of circuits and behavior. In this review, we discuss the molecular profiling of cell types in circuits underlying social behaviors in mice as a prominent case study. We first highlight key roles of molecularly identified sensory and downstream neurons involved in sexually dimorphic behaviors. We then propose future opportunities to define cell types using multimodal criteria, especially gene expression, physiology, as well as the developmental origin, to advance our understanding of these circuits.

Multisensory Logic of Infant-Directed Aggression by Males.

Newborn mice emit signals that promote parenting from mothers and fathers but trigger aggressive responses from virgin males. Although pup-directed attacks by males require vomeronasal function, the specific infant cues that elicit this behavior are unknown. We developed a behavioral paradigm based on reconstituted pup cues and showed that discrete infant morphological features combined with salivary chemosignals elicit robust male aggression. Seven vomeronasal receptors were identified based on infant-mediated activity, and the involvement of two receptors, Vmn2r65 and Vmn2r88, in infant-directed aggression was demonstrated by genetic deletion. Using the activation of these receptors as readouts for biochemical fractionation, we isolated two pheromonal compounds, the submandibular gland protein C and hemoglobins. Unexpectedly, none of the identified vomeronasal receptors and associated cues were specific to pups. Thus, infant-mediated aggression by virgin males relies on the recognition of pup's physical traits in addition to parental and infant chemical cues.

Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy.

Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution.

Dopamine neurons projecting to the posterior striatum form an anatomically distinct subclass.

Combining rabies-virus tracing, optical clearing (CLARITY), and whole-brain light-sheet imaging, we mapped the monosynaptic inputs to midbrain dopamine neurons projecting to different targets (different parts of the striatum, cortex, amygdala, etc) in mice. We found that most populations of dopamine neurons receive a similar set of inputs rather than forming strong reciprocal connections with their target areas. A common feature among most populations of dopamine neurons was the existence of dense 'clusters' of inputs within the ventral striatum. However, we found that dopamine neurons projecting to the posterior striatum were outliers, receiving relatively few inputs from the ventral striatum and instead receiving more inputs from the globus pallidus, subthalamic nucleus, and zona incerta. These results lay a foundation for understanding the input/output structure of the midbrain dopamine circuit and demonstrate that dopamine neurons projecting to the posterior striatum constitute a unique class of dopamine neurons regulated by different inputs.

Molecular organization of vomeronasal chemoreception.

The vomeronasal organ (VNO) has a key role in mediating the social and defensive responses of many terrestrial vertebrates to species- and sex-specific chemosignals. More than 250 putative pheromone receptors have been identified in the mouse VNO, but the nature of the signals detected by individual VNO receptors has not yet been elucidated. To gain insight into the molecular logic of VNO detection leading to mating, aggression or defensive responses, we sought to uncover the response profiles of individual vomeronasal receptors to a wide range of animal cues. Here we describe the repertoire of behaviourally and physiologically relevant stimuli detected by a large number of individual vomeronasal receptors in mice, and define a global map of vomeronasal signal detection. We demonstrate that the two classes (V1R and V2R) of vomeronasal receptors use fundamentally different strategies to encode chemosensory information, and that distinct receptor subfamilies have evolved towards the specific recognition of certain animal groups or chemical structures. The association of large subsets of vomeronasal receptors with cognate, ethologically and physiologically relevant stimuli establishes the molecular foundation of vomeronasal information coding, and opens new avenues for further investigating the neural mechanisms underlying behaviour specificity.

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans.

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Transcription of histone gene cluster by differential core-promoter factors.

The 100 copies of tandemly arrayed Drosophila linker (H1) and core (H2A/B and H3/H4) histone gene cluster are coordinately regulated during the cell cycle. However, the molecular mechanisms that must allow differential transcription of linker versus core histones prevalent during development remain elusive. Here, we used fluorescence imaging, biochemistry, and genetics to show that TBP (TATA-box-binding protein)-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription. Importantly, TRF2-depleted polytene chromosomes display severe chromosomal structural defects. This selective usage of TRF2 and TBP provides a novel mechanism to differentially direct transcription within the histone cluster. Moreover, genome-wide chromatin immunoprecipitation (ChIP)-on-chip analyses coupled with RNA interference (RNAi)-mediated functional studies revealed that TRF2 targets several classes of TATA-less promoters of >1000 genes including those driving transcription of essential chromatin organization and protein synthesis genes. Our studies establish that TRF2 promoter recognition complexes play a significantly more central role in governing metazoan transcription than previously appreciated.

Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription.

Metazoans have evolved multiple paralogues of the TATA binding protein (TBP), adding another tunable level of gene control at core promoters. While TBP-related factor 1 (TRF1) shares extensive homology with TBP and can direct both Pol II and Pol III transcription in vitro, TRF1 target sites in vivo have remained elusive. Here, we report the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. We found 354 TRF1-binding sites genome-wide with approximately 78% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol III-dependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. We provide direct evidence that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). Our studies suggest that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila and possibly other insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription.

Coactivator cross-talk specifies transcriptional output.

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. Here we have used RNA interference (RNAi) in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. We exploited the robust response of the metallothionein genes to heavy metal as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, we find that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a "checkpoint complex" for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, we find that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, we find that the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation.

Targeting genes and transcription factors to segregated nuclear compartments.

With increasingly detailed images of nuclear structures revealed by advanced microscopy, a remarkably compartmentalized cell nucleus has come into focus. Although this complex nuclear organization remains largely unexplored, some progress has been made in deciphering the functional aspects of various subnuclear structures, revealing how this elaborate framework can influence gene activation. Several recent studies have helped illustrate how cells might utilize the nuclear architecture as an additional level of transcriptional control, perhaps by targeting genes and regulatory factors to specific sites within the nucleus that are designated for active RNA synthesis.